This is a continuation of international application Serial No. PCT/CA98/00762, filed Aug. 7, 1998, the entire text of which is incorporated herein by reference.
The present invention relates to a system for creating digital images of fluorescent, luminescent, or bright field specimens. The system is flexible, both in its mode of illumination and in that specimens may be arrayed in matrices (e.g. well plates) or randomly arranged (e.g. chemiluminescent colonies, gel media).
The present invention is primarily an assay imaging system. An assay may be defined as a measurement of physical properties (chemical, biochemical, physiological or other) within a specimen. Assays are used, typically, in the areas of molecular biology, genomics, and pharmaceutics.
The standard assay specimen container is a plastic or glass plate containing 96 small chambers, termed wells. Detection instruments and robotic plate handling technologies have evolved to make efficient use of the 96 well plate, and to provide as high a throughput as possible with this plate format. Current screening technologies can allow a large screening laboratory using 96 well plates to process some thousands of compounds per day.
Recently, there has been a massive growth in the number of compounds available for testing. In part, this is due to an increased exploitation of biodiversity resources to generate natural compounds. In greater part, the proliferation of new compounds is a result of a new chemical technology, termed combinatorial chemistry. In combinatorial chemistry, large numbers of related compounds are synthesized (using permutations of chemical building blocks), and must then be tested for medicinal value.
With new discoveries in natural compounds, and with the advent of combinatorial chemistry, pharmaceutical companies and biotechnology companies are generating extensive xe2x80x9clibrariesxe2x80x9d of untested compounds. These libraries can easily contain millions of compounds. Standard measurement technologies cannot cope with the volume, and new technologies are needed which will increase the rate at which compounds can undergo initial testing (screening) for medicinal value. To show a major advantage over standard technologies, new technologies should allow throughput to exceed 100,000 compounds/instrument per day. Imaging technologies have the promise to increase throughput to the required levels. They have the additional advantage of flexibility, in that an imaging system can be applied to assays in wells and other formats, and to assays which are static or which change over time.
Most assays are designed so that changes in the absorbance, transmission, or emission of light reflect reactions within the specimen. Therefore, most assay measurement instruments detect alterations in luminance as their operating principle. For detection instruments, bioluminescence or chemiluminescence provides the simplest type of assay, in that there is no need to apply illumination. Absorbance assays involve transilluminating the specimen, usually with monochromatic light. The reaction of interest affects the extent to which the light is absorbed by a fluid, and this absorbance may be measured.
Fluorescence is emitted when a fluorophore interacts with an incident photon (excitation). Absorption of the photon causes an electron in the fluorophore to rise from its ground state to a higher energy level. Then, the electron reverts to its original level, releasing a photon (fluorescence emission) hose wavelength depends upon the amount of energy that is released during reversion. A given fluorophore may emit at single or multiple wavelengths (creating an emission spectrum), as electrons drop from various orbitals to their ground states. The emission spectrum is constant for each species of fluorophore.
Fluorescence assays require the application of an intense monochromatic illumination beam, termed xe2x80x9cexcitationxe2x80x9d. Fluorescence assays are used for the following types of applications:
1. An detector tuned to a specific emission spectrum can be used to localize a fluorophore. For example, wells which contain cells expressing a fluorescently tagged protein can be discriminated from wells which do not.
2. By measuring the intensity of fluorescence, an detector system can estimate the concentration of a fluorescent molecule.
3. Changes in the fluorophore molecule (such as binding of fura-2 to Ca++) will lead to alterations in the emission spectrum. A detector can be used to measure these spectral changes, as an indication of changes in the environment of the fluorophore.
Wells
Each well contains a discrete condition of the experiment, and alterations in light emission are measured to determine whether that condition yields favorable properties. xe2x80x9cWell platexe2x80x9d assays are higher in throughput and lower in cost than similar assays in discrete containers.
Reactions within the wells may be of many kinds. In chemistry assays, molecules of different compounds (e.g. a drug candidate and a receptor molecule) are placed into the same well, and the interaction between those compounds is observed. In cell-based assays, each well contains a population of living cells, and effects of compounds on these cells are observed.
Most assays are conducted by making a single measurement from each well. However, it is also possible to record changes over time, by measuring each well repeatedly. The use of repeated observations could be termed a xe2x80x9cdynamicxe2x80x9d assay.
Standard well plates contain 96 or 384 wells in an area of about 8xc3x9712 cm. The trend is towards miniaturization of the wells. Prototypes containing 864 wells or more are under evaluation at many sites. The goal is to develop plates with high density arrays of small xe2x80x9cmicrowellsxe2x80x9d (e.g. thousands/plate) with small fill volumes. That is, miniaturized wells might contain 1 ul of fluid instead of the 100 ul or more used in a typical 96 or 384 well assay.
Miniaturized assay formats promise to achieve dramatic cost reductions and to simplify disposal procedures, while allowing many more assays to be conducted.
Hybridization Arrays and Genetic Assays
Low throughput methods of genetic analysis use various electrophoretic procedures. Methods for increasing throughput and decreasing costs of genomic assays include arraying DNA clones (cDNAs) or synthetic oligonucleotides onto flat support membranes or slips of treated glass. The arrays of cDNAs or oligonucleotides (termed high density grids) are then hybridized to samples of genomic material to quantify levels of gene expression, or to localize relevant sequences. In the past, most hybridization assays have been conducted using isotopic label and storage phosphor imaging systems for detection. However, nonisotopic methods (particularly fluorescence) are under investigation in many laboratories.
Nonisotopic high density grids provide the potential for very high throughput at low cost, and various detection technologies are being developed for these specimens.
Free Format Assays
Assays which occur within a regularly spaced array (wells, cDNAs within a grid) can be referred to as fixed format assays. Specimens that are irregularly distributed can be termed free format assays. Examples of free format assays include electrophoregrams, bacterial colonies in culture, and various combinatorial assays in which bead-bound compounds are distributed over a tissue culture. The common factor in these free format assays is that areas of altered luminance can occur at any spatial location.
Instruments designed for fixed format assays (fluorescence plate readers, liquid scintillation counters, etc.) only read from defined locations in the specimen. They are not useful with specimens in which effects lie at locations that are not predefined. In contrast, imaging systems are able to detect and quantify reactions at any position within an image, and there is an extensive history of image analysis being applied to free format assays.
Summary of Types of Assays
Pharmaceutical companies are faced with unprecedented numbers of new compounds, which must be screened for medicinal value. Specimens are screened using luminescence, absorbance, or fluorescence technologies. Specimen formats include standard and miniaturized well plates, high density arrays of hybridization assays, and free format assays. The present invention is designed to be useful for all of these technologies and specimen formats.
Area Imaging Systems
An area imaging system places the entire specimen onto a detector plane at one time. There is no need to move PMTs or to scan a laser, because the camera images the entire specimen onto many small detector elements (usually CCDs), in parallel. The parallel acquisition phase is followed by a reading out of the entire image from the detector. Readout is a serial process, but is relatively fast, with rates ranging from thousands to millions of pixels/second.
Area imaging systems offer some very attractive potential advantages:
1. Because the entire specimen is imaged at once, the detection process can be very quick.
2. It is relatively easy to acquire a timed series of images for dynamic assays.
3. Given an appropriate illumination system, any excitation wavelength can be applied.
4. Luminescence reactions (bioluminescence, chemiluminescence) can be imaged.
5. Free or fixed format specimens can be imaged.
In many applications (e.g. film autoradiography), area imaging has a history, instrument developers have experience, and the problems are well understood. In contrast, assay imaging presents new and significant technical challenges. There is no established practice for imaging assays, and very few instrument developers have practical experience with both area imaging and assay technologies.
Luminescence Area Imaging
We will define luminescence as light emitted from a specimen, without excitation by external light. Most luminescence reactions are quite dim, and this can make extreme demands upon detection. The standard area imaging strategy is to use scientific grade CCD cameras, which achieve relatively high levels of sensitivity. Intensified CCD cameras have also been used.
Typical prior art applies area imaging to luminescent assays on flat membranes. Descriptions of well imaging are flawed in that no correction for parallax error is described. Telecentric lenses have not been used, because prior art telecentric lenses have not been able to collect enough light.
Prior art also fails to disclose the use of software that would allow automated analysis of targets which do not lie in a perfectly regular array, or software which models variations in measurement efficiency from well to well.
The novel features of the present invention (efficient light collection combined with freedom from parallax, target detection software, calibration software which minimizes variations in measurement efficiency) allow it to be used with luminescent specimens that lie beyond the capabilities of prior art luminescence imaging systems.
Fluorescence Area Imaging
Area imaging is in routine use for fluorescence microscopy, where epifluorescence is the universal method and is easily applied. On a microscope, epifluorescence is implemented via an arrangement incorporating a dichroic mirror and interference filters. Prior art in fluorescence microscopy is irrelevant to the present invention, which is specifically designed for macro specimens.
Macro-level, area fluorescence imaging is routinely applied with gels and blots. Although some specialized research systems have been reported, prior art is dominated by low cost commercial systems for routine gel/blot fluorescence. Typically, this type of imaging has been compromised by sub-optimal delivery of excitation illumination. The most common form of area illumination uses gas discharge illuminators (e.g. UV light boxes). The lamps are coated with a filter that limits emission to the peaks emitted by the excited gas within the lamp. Thus, most prior art systems offer a limited number of excitation wavelengths.
Many prior art systems, as those using transilluminators, pass excitation directly through the specimen and into the collection optics. For the highest sensitivity, however, it is important that excitation not be delivered directly into the detection optics. With direct delivery, both the excitation and the emission wavelength are detected, with the excitation at much higher levels than the emission. The excitation wavelength can be filtered, selectively, but enough excitation remains to degrade sensitivity.
To avoid the direct detection of excitation, some prior art systems deliver excitation from above, from the side, via dark field, or using refraction (e.g. evanescent wave) into the specimen. In these cases, the detection optics view fluorescence emission from the specimen, with minimal detection of excitation. However, these techniques have severe limitations. Side-mounted fiber optic illuminators deliver uneven light, and can only be used with flat specimens. When they are used with wells, light enters the specimen at an angle and fails to penetrate deeper regions. Similarly, refractive or dark field illuminators do not penetrate into deep wells, cannot be used with opaque specimens, and tend to provide rather dim levels of illumination.
We can summarize the characteristics of prior art systems as follows:
1. Most are limited to the wavelengths emitted by gas discharge lamps. Typically some combination of UVA, UVB, UVC, and/or white light lamps is provided. Other wavelengths cannot be obtained, and this is a sever disadvantage.
2. Wavelengths cannot be altered during an assay. If the illumination must be changed during the assay (e.g. as for calcium measurement with fura-2), the devices cannot be adapted.
3. Insensitive to small alterations in fluorescence. Transillumination comes from directly below the specimen into the detector optics. Therefore, even very good filters fail to remove all of the incident illumination, and this creates a high background of nonspecific illumination. Small alterations in fluorescence (typical of many assays) are lost within the nonspecific background.
4. Inefficient cameras and lenses. A very few systems use high-performance cameras. Even these few systems use standard CCTV or photographic lenses, and can only be applied to bright specimens.
5. Parallax error precludes accurate well imaging. As wide-aperture, telecentric lenses have not been available, these systems exhibit parallax error when imaging wells.
In one example of a prior art fluorescence system, a fiber optic is coupled to the specimen on its input, and to a CCD camera on its output. This use of a fiber optic lens in the input to a camera yields freedom from parallax error and efficient light collection. The CCD camera can be run in photon counting mode to obtain single photon detection capability. In achieving parallax-free images and high sensitivity to light, the fiber-coupled camera is like the present invention. However, fiber coupling to the specimen has the following major disadvantages:
1. A fiber-coupled system is limited to specimens that are transilluminated, because there is no place to insert an epi-illumination mechanism. Therefore, the fiber-coupled system would not be as sensitive to subtle variations in fluoresence as is the present invention (epi-illumination).
2. Because fiber optics have a very shallow depth of field (typically about 1 xcexcm), the fiber-coupled system must view the specimen through a thin, clear film of glass, plastic, or other material. Not all assays can be conducted in plates having thin, clear bottoms. If assays must be conducted in opaque plates, the fiber-coupled system becomes inefficient, because it can not be brought close enough to the fluid within the wells to achieve focus.
3. Because of its limited depth of field, a fiber-coupled optic exhibits compromised performance with deep specimens. For example, fluid in well plates is typically more than 1 mm deep. Only a small part of the fluid is in focus with a fiber-coupled optic. This leads to inefficient light collection from those parts of the fluid that do not lie in critical focus.
4. The fiber coupled system could not be used for fluorescence polarization studies, because of the effects (birefringence, etc.) of the fiber on polarized light.
5. Well plates are 8xc3x9712 cm. Image forming fiber optics of this size are very difficult and expensive to construct, and must be brought to a much smaller CCD by tapering the fiber bundle into a minified configuration. Minification results in major losses in transmission efficiency, which might drop from 70% to 10%. To avoid both the high cost of a large fiber optic, and the loss of transmission efficiency resulting from minification, the specimen would have to be acquired as a number of subimages. In this case, a translation table moves the specimen or camera, and a straight or slightly minified fiber bundle of reasonable size is used. Following a scan, the subimages would be reassembled to show the entire specimen. This multiple acquisition is slow, affects accuracy and precludes use of the device with assays which change over time.
Other examples of prior art fluorescence systems include use of a cooled CCD camera, with an illuminator that is mounted laterally, or above the specimen. In some cases, an excitation filter wheel can be inserted prior to a fiber optic light delivery system, to achieve some of the functions of the present invention (selection of wavelengths). These prior art systems all have the following major disadvantages:
1. A side or top-mounted illuminator results in very uneven illumination with almost any specimen, and is completely unsuitable for use with wells.
2. Systems lack a telecentric lens, so parallax would preclude imaging of assays in wells.
3. Target detection software is lacking.
There is another prior art fluorescence system (Fluorescence Imaging Plate Readerxe2x80x94FLIPR, Molecular Devices) which uses laser illumination applied at an angle to the wells in a 96 well plate. A pinhole mask is placed over the wells, so that the angled light only reaches a small part of the specimen fluid at the bottom of each well. Data are detected using a cooled CCD camera. FLIPR is inflexible, in that it runs only in 96 well format, uses specific laser lines, does not offer epi-illumination, cannot illuminate the entire fluid contents of deep wells, and is not applicable to luminescence imaging, free format imaging, or higher density well formulations.
Science Applications International Corporation (SAIC, San Diego, Calif) was developing the RTFluorimeter, a high throughput fluorescence screening system for kinetic assays in 96 well format. The RTFluorimeter includes a robot for well filling, a high power arc lamp, a filter wheel for excitation wavelength selection, and a CCD array detector. The system illuminated using angled fiber optics placed at the sides of the specimen, and did not include epi-illumination. Performance data and specific imaging design information have not been forthcoming, and this system has been discontinued.
There is extensive prior art in the use of imaging to detect fluorescence assays incorporated within microfabricated devices (often termed xe2x80x9cgenosensorsxe2x80x9d). Some genosensors use scanning imagers (e.g. the Molecular Dynamics or Hewlett Packard genechip readers), and detect light with a photomultiplier. Others use area CCDs to detect alterations at assay sites fabricated directly onto the CCD, or onto a coverslip that can be placed on the CCD. It can be envisaged that many other types of genosensors will appear in the near future.
Macro Fluorescence
Novel features of the present invention minimize the disadvantages of prior art macro fluorescence systems. These features include the following:
1. The device can accept light from almost any source. Therefore, illumination wavelengths may be selected without regard to the peak(s) of a gas discharge lamp or laser.
2. Using a computer-controlled filter wheel or other device, illumination may be altered during an assay,
3. Small alterations in fluorescence emission can be detected, because fluorescence illumination comes via epi-illumination. Direct excitation illumination does not enter the optics, and the nonspecific background is as low as possible.
4. The camera and lens form a highly optimized system for use with dim specimens.
5. The telecentric lens removes parallax error so well plate assays are accurate.
6. The optics can be adapted to many forms of fluorescence, including time resolved and fluorescence polarization assays.
7. The entire fluorescence illumination and detection system is integrated within a single mechanical assembly. This assembly is easy to integrate with automated specimen handling equipment.
For fluorescence, a critical advantage of the present invention is its epi-illuminating lens. It is flexible in application, allows simultaneous imaging of the entire specimen, illuminates, collects data without high levels of background or parallax error, is easily integrated with specimen handling automation, and is usable with dim specimens.
In one particular, adaptability to opaque media, the lens of the present invention minimizes the disadvantages of a fiber-coupled optic. The lens focuses some distance away from the specimen, uses epi-illumination, and has large depth of field. These features allow the focusing of fluids lying within opaque media and, more generally, focusing of fluids of typical depth (e.g. 1 mm). Another advantage of the present invention is that, as there is no fiber optic lens to act as a light scramble, fluorescence polarization assays may be performed. Finally, the lens of the present invention images the entire well plate in a single acquisition. This avoids the need for translation tables, and makes both accurate calibration and dynamic assays much easier to perform.
Genomic Assays
In one particular, the present invention has advantages for genomic assays. Genomic assays are in flux, and many labeling methods (fluorescence, luminescence, absorbance, isotopic) and formats are likely to appear. Unlike the scanning imagers currently used for these genomic assays, the present invention allows almost any specimen format (including wells), any fluorescence wavelength, and is capable of luminescence detection. Luminescence methods can offer the highest levels of sensitivity in some genomic assays.
The flexibility of the present invention is unique, and makes it suitable as a single tool for laboratories performing genomic assay prototyping. In assay prototyping, is can be difficult to predict the specific wavelengths and specimen formats that will be used. The present invention allows a laboratory to construct an assay using almost any labeling and detection paradigm. There is no need to select from the small number of labeling and detection methods supported by a specific laser scanner.
The present invention could also be used for mass screening (using high-volume fabrication), when scanning laser excitation is inappropriate, or when luminescent detection is required. In sum, it is a purpose of the invention to provide a more flexible alternative to prior art laser scanning of genomic assays.
Summary of Area Fluorescence Imaging
The present invention includes features that are novel, both singly and in combination. These are:
1. macro epi-illumination;
2. ability of a single instrument to conduct both accurate (parallax free, calibrated) fluorescent and non-fluorescent assays;
3. freedom from parallax;
4. easily adapted to fluorescence polarization, time resolved fluorescence, and other methods;
5. easy integration of fluorescence illumination with specimen handling and dispensing automation;
6. software includes calibration methods that minimize errors inherent to area imaging;
7. software finds and quantifies targets.
A system in accordance with the present invention includes a CCD detector, lens, illumination system, imaging system, and software. Together, these components are configured as a system that can be used with fixed or free formats, with wells or flat specimens, and with any form of illumination (fluorescence, luminescence, transmission). The system exhibits measurement performance similar to that of standard, non-imaging measurement instruments, while having the additional advantages of an imaging system.
Major Components of a System in Accordance with the Invention
a) Sensitive CCD Detector
Area imaging systems use CCD arrays to form images. Factors which influence the ability of CCD arrays to detect small numbers of incoming photons include quantum efficiency, readout noise, dark noise, and dynamic range. Preferably, the present invention uses a CCD array with high quantum efficiency, low readout and dark noise, and broad dynamic range.
b) A Lens Which Combines Telecentricity and Large Aperture
The lens of the present invention is unique in that it combines telecentricity with a large aperture. This combination allows accurate reading of dim assays within wells.
c) True Epifluorescence Optics
An epifluorescence optic both illuminates and collects through the same lens. Epifluorescence optics must include a source of excitation, and a barrier filter which removes excitation from the detected signal and leaves only the fluorescence emitted from the specimen. The present invention incorporates both a source of excitation and a barrier filter within its lens.
The epifluorescence optics are beneficial in yielding lower backgrounds, broader dynamic range, and more linear fluorescence response than dorsal, lateral, refractive, or transilluminators. The ability to deliver large area, monochromatic epi-illumination is a unique capability of the present invention.
d) Optional Components for Ultra-Low Light Imaging
Although any area detection technology may be used, the preferred detector is a thinned, back-illuminated and large CCD, which is capable of imaging even low levels of fluorescence and luminescence. For the most extreme low light conditions, as with the scintillation proximity assay, the present invention allows use of a camera cooled to very low temperatures (e.g. the CCD runs at xe2x88x9270 degrees C). Cooling may be by liquid nitrogen, forced probe mechanism, multistage thermoelectric, or any other means. As an alternative, the present invention allows substitution of a fiber optic lens for the epi-illuminating lens. This use of a fiber optic lens sacrifices flexibility and fluorescence capability, in return for higher sensitivity with luminescent assays.
The present invention also allows use of a light amplifier, inserted between the lens and the CCD camera. In one preferred configuration, this light amplifier is an image intensifier.
e) Flexible Illumination Source for Fluorescence Excitation
Application of even illumination of selectable wavelength over an 8xc3x9712 cm area is a feature of the present invention. Illumination is delivered via a standard fiber optic coupling. This allows the use of almost any illumination source (halogen, arc lamp, laser, etc.). In a preferred configuration incorporating a halogen or arc lamp, any wavelength of monochromatic illumination may be selected by precision filters (usually standard interference filters). These are readily available in the small sizes used. An optical coupler/computer-controlled filter wheel is preferred for selection of excitation wavelengths. The output of this device may be taken to a specially designed fiber optic plate for transillumination, to a fiber optic ring, line, or panel light for dorsal illumination, or (preferably) to a fiber optic illumination assembly within the lens, for epi-illumination.
f) Software That Integrates Control, Detection, and Analysis
Area imaging is inherently more complex than either nonimaging detection, or the use of scanning imagers. In particular, nonimaging counting systems have a relatively easy task. They only need to control the scanning process, control internal calibration, and create a small array of data points representing each well. The sequence of steps might be as follows:
1. Calibrate detector against internal standard.
2. Illuminate one well.
3. Position a PMT over the illuminated well.
4. Read well.
5. Transfer data to spreadsheet.
6. Illuminate next well and repeat.
An area imaging system has a much more difficult task. Imaging a well plate might include the following requirements:
1. Provide adequate illumination over the entire plate.
2. Control a high performance camera.
3. Store geometric and density correction factors.
4. Image specimen.
5. Correct geometric and density variation.
6. If necessary, calibrate image to standards within the specimen.
7. Locate each well and quantify intensity, with respect to a calibration function.
8. Transfer data to spreadsheet.
These tasks can only be performed if the imaging system is equipped with software that performs functions 2-8, above. The present invention incorporates such software, including novel software for locating specimens, and for calibrating the imaging system to minimize error.
g) Optional Components for Time Resolved and Polarization Fluorescence
A standard way to minimize background is to take advantage of the long-lived fluorescence properties of specific fluors. With a long-lived fluor, the illumination source can be strobed very rapidly, so that excitation is applied for a brief period, and is then blocked. Fluorescence tends to fade gradually (e.g. over a period of 10 msec after blocking of excitation), so measurement can be made during the excitation blocking period. This measurement after blocking of the excitation illumination should be free of reflections, and of direct contamination by the excitation wavelength. This delayed measurement is known as time-resolved fluorescence (TRF).
The major advantage of TRF is that it results in much lower levels of background fluorescence. The major disadvantages of TRF are that it requires specialized hardware for the rapid cycling of excitation and blocking, and that a limited number of special fluors (typically using Europium, Lanthanide, or Terbium chelates) are suitable for the procedure.
Typically, the present invention is applied to time resolved fluorescence by using both a shutter and a strobe light source. The strobe provides brief and precisely gated periods of illumination. The shutter protects the CCD from exposure while the illumination is on.
Various types of shutter are possible:
An image intensifier is mounted between the epi-illuminating lens and the CCD detector. The intensifier serves to amplify the light coming from the fluor and, by gating, the intensifier serves as a shutter. When the intensifier is gated on, light is allowed to reach the CCD. When the intensifier is gated off, light is prevented from reaching the CCD. Gating can be very quick, so that the intensifier on period coincides with the optimal period of delayed fluorescence from the specimen.
A chopper wheel, liquid crystal, leaf, or other shutter may be mounted within the optical path, after the specimen but prior to the detector. For example, the shutter could be located at the barrier filter position within the lens. The shutter is time-locked to the excitation strobe, so as to achieve the required delay between the on-off cycle of the strobe, and the exposure period of the CCD.
Although the preferred illumination source is a strobe lamp, other forms of illumination (e.g. pulsed laser, pulsed arc lamp, continuous arc lamp) may be envisaged by those skilled in the art. If the illumination cannot be gated precisely, a second shutter mechanism is required to provide a precise on-off cycle.
A critical advantage of the present invention for TRF is that illumination is applied via the telecentric, epi-illuminating lens. This lens provides a convenient means by which fluorescence excitation may be applied, telecentric collection free of parallax, and efficient optics that collect as much as possible of the dim light emitted in delayed fluorescence paradigms.
The lens of the present invention may also be configured for fluorescence polarization studies, by adding a ring polarizer on the output of the internal ring light, and an analyzer into the lens barrel.
Features of the Analysis Software Finding of Targets
An image contains both targets and background. Often, there is some ambiguity about which parts of the image constitute the target component. Extensive prior art deals with the automated extraction of target data from images. The extraction process is known as xe2x80x9csegmentationxe2x80x9d. Typically, segmentation algorithms use density, color, texture, or other image characteristics to define a valid target. Any pixel that meets the target definition criteria is designated as a target pixel.
The present invention performs segmentation of the usual kinds, using any combination of image characteristics, in addition to novel forms of segmentation. The usual type of segmentation is preferable for free format specimens. Typically, targets are defined by their luminance value, and are then detected automatically at any points within the image. For imaging of wells, hybridization arrays, and other specimens in a regularly spaced (fixed) format, the present invention uses a novel form of segmentation which allows very large numbers of targets (e.g. 60,000) to be analyzed, with full automation.
The novel segmentation procedure uses a xe2x80x9cfixed probexe2x80x9d strategy, in which an array of probes of constant size is placed onto the sample. For this purpose, an analogy is made to liquid scintillation counting. In liquid scintillation counting of isotopically labeled specimens it is common to cut out (using a circular punch) pieces of membrane of fixed size. These pieces are placed into vials of scintillation fluid for counting. As a result of the count, the total amount of radioactivity in each sample piece becomes known. Similarly, with the present software, an array of fixed probes is applied to the image, at intervals specified by the grid definition.
Positioning software is required, because the array of computer-generated probes is unlikely to accord exactly with the true locations of all the targets in the specimen. Variation in target position arises because spotting robots are not perfect, plates may vary slightly in position, and so forth. Therefore, prior to quantification, the image is processed and parsed to rationalize the location of each target within the array. Essentially, the algorithm looks for any targets that may be recognized on the basis of distinct densities. It fits these recognizable targets into the most likely locations within the grid, using a fuzzy logic algorithm. Then, it fits the less distinct dots into their most likely locations, using the higher-density dots as anchor points for the alignment.
To the user, the procedure appears automatic. Once the fixed probe array has been defined on the specimen, the computer performs automatic alignment of the array elements to the most appropriate positions in the matrix. Any elements that remain misaligned may be manually edited.
Statistical Segmentation
Luminance data from the aligned matrix are output to software, which creates a distribution of data from every fixed probe. Typically, a mean and standard deviation are calculated for this distribution, and targets may be further defined as xe2x80x9chitsxe2x80x9d using a variance criterion for the distribution. For example, we might define targets that lie more than four standard deviation units from the mean of the distribution as hits. This use of variance-based hit definition is termed xe2x80x9cstatistical segmentationxe2x80x9d.
Statistical segmentation requires that every sample in the matrix be analyzed, even if it is not detectable by standard segmentation methods. Therefore, when used for well plate or other grid assays, statistical segmentation requires the use of aligned, fixed probes as described above.
The Measurement Efficiency Calibration
The present invention implements a novel means by which measurement efficiency may be corrected. This allows correction of variations in collection efficiency of the lens or camera. In practice, the system views a set of reference standards which span all or part of the range of intensities observed in actual specimens. Typically, these standards will be well plates of the same kind, and containing a substance responding to the same wavelengths as are to be used for the assay in question. Preferably, three (or more) plates are made to contain low, medium, and high concentrations of light emitting substance. The use of multiple reference concentrations allows nonlinear variations in measurement efficiency to be accommodated.
Once the reference plates have been viewed, the system can derive discrete models for measurement efficiency at each well. That is, the reference plates allow us to create, at each spatial location in the image, a known relation between the intensity of light observed by the system, and the concentration of light emitting substance that is actually present. The system uses these data to model and correct subsequent measurements obtained from each well. The result is a low level of error variation between wells. Typically, the present invention yields coefficients of variation that are very similar to those obtained from nonimaging counting systems. This use of a correction for well-to-well variation in measurement efficiency is highly advantageous, if an area imaging system is to achieve acceptable accuracy in quantifying wells.